Synthesis of tantalum sol-gel and production method of microextraction syringe for the purpose of enrichment of phosphopeptides

ABSTRACT

The present invention relates to a production method of sol-gel ( 100 ) comprising tantalum metal and a method ( 200 ) concerning use of the sol-gel produced for enrichment of phosphopeptides by means of a microextraction syringe. The syringe which is filled with the sol-gel comprising tantalum metal enables to make analysis of natural samples, that comprise low amount of phosphoprotein, with high precision and in a very short time and it shows compliance with mass spectrometric techniques. Thus, it can be used for application of fast and practical analytical methods for the analysis made in phosphoproteomics studies wherein mass spectrometric techniques are used.

CROSS REFERENCE TO THE RELATED APPLICATION

This application claims the benefit to Turkish Patent Application No2013/14066 filed Dec. 3, 2013, the content of which is incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to a production method of sol-gelcomprising tantalum metal and a method concerning use of the sol-gelproduced for enrichment of phosphopeptides by means of a microextractionsyringe.

BACKGROUND OF THE INVENTION

In order to increase enrichment of phosphopeptides and their assaysensitivities, various techniques are developed with each passing day.Methods based on covalent bonding or non-covalent interactions are usedwith the purpose of enriching phosphopeptides. However, use ofenrichment methods based on non-covalent interactions is more common. Inorder to analyse phosphopeptides, which are included in a naturalsample, by means of mass spectrometric techniques with high selectivityand sensitivity it is aimed to develop practical phosphopeptideenrichment methods wherein reusable and economical materials are used.By means of using these methods and materials, it is contributed tomedical applications aiming to create diagnosis and treatment methods ofdiseases such as cancer or Alzheimer's disease occurring as a result ofbasic cellular activity disorders.

In commonly used phosphopeptide enrichment methods, principle ofinteractions of phosphate groups on peptides with their metal oxides isutilized. Purification studies can be done with chromatographic methodsby taking advantage of negatively-charged phosphate groups included instructures of phosphopeptides. Particularly, affinity chromatographymethods based on principle of interactions of negatively-chargedphosphate groups with their metal cations is frequently used. There aremany different chromatographic methods wherein metals, developed withthis approach, such as iron (LI-YUAN, 2010), gallium (SALIM, 2012) ormetal oxides such as titanium dioxide (KRENKOVA 2013), zirconium dioxide(NELSON, 2010) are used

Materials such as titanium dioxide (TiO₂) and zirconium dioxide (ZrO₂)also become suitable for binding of phosphopeptides at low pH values. Bymeans of the studies done it is shown that binding affinity of ZrO₂ tophosphate group is a lot more than affinity thereof to carboxylateanions (BLACKWELL, 1991). In addition, ZrO₂ has been used as achromatographic material for a long time because of its physical andthermal stability. Using ZrO₂ surfaces having these features is suitablefor studies of phosphopeptide enrichment. Although TiO₂ is the mostcommonly used materials of phosphopeptide enrichment on metal oxidesurface, oxides of other various materials other than titanium are alsoused in studies of phosphopeptide enrichment. Fe₃O₄-coated AI₂O₃ (LI,2007) or Ga₂O₃ (LI, 2008) magnetic microspheres were used in studies ofphosphopeptide enrichment and successful results were obtained. Also, itwas shown that SnO₂ (STURM, 2008) and Nb₂O₅ (FICARRO, 2008) metal oxidescan be used in phosphopeptide enrichment.

Sol-gel materials attract attention due to the fact that they can beproduced as having pre determinable pore size and shape; and because oftheir potentials in separations, adsorption, catalysis, drug release andchemical sensor applications needed in molecular identification. itcannot be said that amorphous metal oxide sol-gel materials are not assuccessful as acrylic-based pressed polymers in chemical applications.However, sol-gel materials have many advantages in comparison to organicpolymers. For example, thermal stability of organic polymers is as lowas it cannot be compared to sol-gels. In sol-gels, control of thickness,porosity and surface area is easy; selectivity and diffusion is alsobetter than acrylic-based polymers (MARX, 2001; LAHAV, 2001).

Phosphoproteom analysis made with mass spectrometric methods are usuallyperformed at two steps. The phosphoprotein to be analyzed isenzymatically disintegrated at first step whereas peptide unitsoccurring as a result of enzymatic digestion are analyzed at the secondstep. Although this method appears to be useful, it may be confrontedwith some problems in applications wherein this approach is used. Thefact that signals belonging to all of phosphopeptides in the medium inthe peptide mass maps obtained cannot be observed, ion formationdecreases as a result of the phosphate group increasing the acidity,other peptide ions in the medium suppress signals belonging tophosphopeptides, phosphopeptides with hydrophilic feature cannot usuallyhold on to column materials (reverse phase) used for purification ofpeptides sufficiently can be given as an example to these problems.

In order to overcome the difficulties encountered in phosphoprotein andphosphopeptide analysis which are made with mass spectrometric methods,it is aimed to develop selective enrichment methods. Enrichment processenables to make phosphopeptide analysis by eliminating the problem ofion suppression arising from existence of peptides not includingphosphate group.

There is no enrichment or identification method which completely showscompliance for the whole of phosphoproteomics studies. In studies doneat the present time, enrichment and purification methods ofphosphoproteins or phosphopeptides must be selected depending on type ofthe sample to be analyzed. In fact currently available methods aresuccessful at enrichment studies of a part of phosphoproteins in theirway. It is considered that development of different and new methodsshowing compliance for each sample in this field will provide quitesubstantial benefit for proteomics studies.

An increase can be observed in amount of impurity in the enrichmentmedium depending on separation of metal ions from the material whereinthey are included, in studies where materials including the metal ionsare present with the purpose of phosphopeptide enrichment. In such acase, efficiency of purification considerably decreases due to the factthat the material used for enrichment is not stable.

Acidic peptides, which are included in the medium apart fromphosphopeptides, may show interest to surfaces of metal oxidesfrequently used in studies of phosphopeptide enrichment such as TiO₂.Extra methods and Chemical additives may be used with the purpose ofremoving other acidic peptides and increasing selectivity of theenrichment method. Separation principles of anion or cation exchangematerials used in enrichment of phosphopeptide are based onelectrostatic interactions between types. Electrostatic interactions arerandom interactions selectivity of which is quite low. Even if typeshaving opposite electrical charge are random they may tend to gettogether with such interactions. It is quite difficult to purify onlysmall amount of phosphopeptides from among quite complex samples such ascell lysate, using such materials. Because of these reasons; durable,inexpensive, reusable phosphopeptide with high-selectivity can bealternative to enrichment methods wherein such materials are used.

The International Patent Document No. WO2012079549 A3 discloses thatmaterials which can be used for the purpose of enrichment ofphosphopeptide by performing surface modification with organometalliccompounds comprising elements of 4B group such as zirconium, hafnium,titanium are developed.

The United States Patent Document No. U.S.20100125128 A1 presentsceramic hydroxyapatite material and analytical method which can be usedin phosphopeptide enrichment applications. It is stated thatphosphopeptides having different features can be separate from eachother besides enrichment of phosphopeptide by means of this method.

The International Patent Document No. WO2010045710 A1 states thatphosphorylated biomolecules such as phosphopeptides and phosphoproteinscan be purified from the medium wherein they exist using an affinitymatrix with metal ion content.

The United States Patent Document No. U.S.8003340 B2 proposes apurification process which is carried out based on the principle ofprecipitation of metal-phosphoprotein complexes obtained as a result ofbinding calcium, barium, molybdenum, cobalt ions to phosphoproteinsselectively.

SUMMARY OF THE INVENTION

An objective of the present invention is to realize a production methodof sol-gel material which enables to make analysis of natural samples,that comprise phosphoproteins or phosphopeptides low concentration, of asyringe filled with sol-gel comprising a tantalum metal with highprecision and in a very short time and works in line with massspectrometric techniques; and a method for use of sol-gel material inenrichment of phosphopeptide.

Another objective of the present invention is to realize a productionmethod of sol-gel material wherein the solid-phase microextractionmaterial, that is developed using the sol-gel synthesized, is reusable;and a method for use of sol-gel material in enrichment ofphosphopeptide.

A further objective of the present invention is to realize a productionmethod of sol-gel material wherein a sol-gel with a content of tantalummetal having chemical stability, quite durable physically and suitablefor use in natural sample analysis is produced; and a method for use ofsol-gel material in enrichment of phosphopeptide.

A yet further objective of the present invention is to realize aproduction method of sol-gel material wherein the sol-gel with thecontent of tantalum metal synthesized is used without being subjected tocalcination process at high temperature; and a method for use of sol-gelmaterial in enrichment of phosphopeptide.

A production method of sol-gel and a method for use of sol-gel inenrichment of phosphopeptide realized to fulfil the objectives of thepresent invention are shown in the figures attached.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of the flow chart concerning the inventive productionmethod of sol-gel.

FIG. 2 is a view of the flow chart of the method for use of theinventive sol-gel in enrichment of phosphopeptide.

FIG. 3 is a mass spectrum of a milk sample form before (A) and after (B)the process of phosphopeptide enrichment is applied, which is generatedas a result of analysis thereof with MALDI-MS (Matrix-assisted LaserDesorption/Ionization-Mass Spectrometry).

FIG. 4 is a view of the sol-gel network structure comprising tantalum.

DETAILED DESCRIPTION OF THE INVENTION

The inventive production method of sol-gel (100) comprises steps of:

-   -   adding one or more of tantalum (V) alkoxide, tantalum (V)        methoxide, tantalum (V) ethoxide, tantalum (V) propoxide,        tantalum (V) isopropoxide and tantalum (V) butoxide into the        mixture (101);    -   performing a mixing process during addition of material (102);    -   adding an acid solution such as HNO₃, HCl, H₂SO₄, CH₃COOH,        CF₃COOH etc. during the mixing process (103);    -   decomposing the sol-gel with tantalum content through a        centrifuge process (104).

In the inventive method (100) before the addition of tantalum metal intothe mixture (101), the solution comprising polyethylene glycol (PEG)(Mr-600-3000) in a ratio of 30-70% by volume is diluted with ethanol:water mixture (18:0.5-18:3.0, v/v) (1%, v/v). After the dilutionprocess, one or more of tantalum (V) alkoxide, tantalum (V) methoxide,tantalum (V) ethoxide, tantalum (V) propoxide, tantalum (V) isopropoxideand tantalum (V) butoxide is/are added into the mixture (101). Duringaddition of starting material, mixing process is carried on (102)quickly. While the mixing process continues, up to 0.1-0.5% of aconcentrated acid solution such as HNO₃, HCl, H₂SO₄, CH₃COOH, CF₃COOH,etc. is added into the synthesis medium (103). Upon addition of acidduring this process carried out at room temperature, it is observed thatsol-gel particles with tantalum content occur (104).

In a preferred embodiment of the invention, the sol-gel with tantalumcontent obtained is washed and then taken into its final form afterbeing dried. The sol-gel with tantalum content is washed with solventssuch as ethanol, water and methanol in the preferred embodiment. Dryingprocess is carried out at temperature of 40° C. in a vacuum environmentbetween 2-200 mbars.

Whereas the method of phosphopeptide enrichment (200) which is realizedusing the sol-gel obtained comprises steps of:

-   -   inserting the sol-gel comprising the tantalum metal into the        syringe (201);    -   adding some acid into the peptide mixture and adjusting PH        (202);    -   drawing a certain amount of the solution generated into the        syringe and releasing it (203);    -   carrying out the washing process (204); and    -   withdrawing the phosphopeptides from the syringe (205).

In the inventive enrichment method (200), after the sot-gel comprisingthe tantalum metal is inserted the into the syringe (201) firstly somemixture of acetonitrile:water:trifluoroacetic acid (1:1:0.005-0.3,v/v/v) is added into the peptide mixture Obtained as a result ofenzymatic digestion and the PH is adjusted (202). PH value of the acidmixture is approximately between 1.0-3.0. In a preferred embodiment, 50μL of the solution generated is drawn into the syringe and released(203). The said process is repeated for 2-6 times. And in order toremove impurities and peptides except phosphopeptides, the mixture ofacetonitrile:water:trifluoroacetic acid (1:11:0.005-0.3, v/v/v) is drawninto the syringe and released. The said is also repeated for 2-6 times.The solution of 10-100 mM ammonium bicarbonate comprising ammonia in thewashing process is pulled through the syringe (204) lastly and thephosphopeptides are withdrawn from the syringe (205).

The microextraction syringe which is filled with the sol-gel comprisingtantalum metal enables to make analysis of natural samples, thatcomprise low amount of phosphoprotein, with high precision and in a veryshort time and it shows compliance with mass spectrometric techniques.Thus, it can be used for application of fast and practical analyticalmethods for the analysis made in phosphoproteomics studies wherein massspectrometric techniques are used. The sot-gel synthesized has physicaland chemical properties which will show compliance to ambient conditionsin terms of being usable in methods of phosphopeptide enrichment. Thesolid-phase microextraction material which is developed using thesol-gel synthesized with the trials made is reusable. The sol-gelcomprising tantalum metal has chemical stability, is quite durablephysically and suitable for use in natural sample analysis.

The sol-gel comprising the tantalum metal synthesized can he usedwithout being subjected to calcination process, which is defined asdigestion of carbonates and hydrates with temperature effectin order toobtain oxide components, at high temperature. The process ofphosphopeptide enrichment is realized over interactions ofphosphopeptides with positive metal centers on the sol-gel surfacecomprising tantalum of the electron-rich phosphate groups. Therefore,phosphopeptides are expected to be negatively charged and the tantalumcenters on the surface are expected to be positively charged during theinteraction between the said types. Electron need of metal on thesurface is provided by the oxygen atoms due to the more regularstructure which is composed as a result of the oxidation occurring bycalcination of the sol-gel surface at high temperature. Therefore,phosphopeptides' interests for sol-gel surface decrease. However, it isconsidered that using sol-gel after steps of synthesis and washing stepsrequired will increase efficiency of the process of phosphopeptideenrichment. The sol-gel which comprises tantalum metal used withoutapplying any calcination process, enables to obtain quite successfulresults at the step of phosphopeptide enrichment.

With the purpose of testing efficiency of the sol-gel obtained in theprocess of phosphopeptide enrichment, besides many mixtures cow's milkshowing effect of different natural sample and high matrix is used.Proteins involved in milk are denaturated before the enrichment processand broken into peptide fragments by the trypsin enzyme. As a result ofthis process, it is ensured that phosphopeptides are separatedselectively using the complex peptide mixture obtained and the syringedeveloped from within matrix.

When the milk sample is analysed with the MALDI-MS (Matrix-assistedLaser Desorption/Ionization-Mass Spectrometry) directly, the massspectrum given in the FIG. 3 is obtained (A). No signal of thephosphopeptides can be observed in this mass spectrum. After the processof phosphopeptide enrichment is realized using the syringe, when thesample is analysed only the mass spectrum wherein the signals of thephosphopeptides are located is obtained (B). This data indicates thatphosphopeptides are separated from the other peptides and impurities inthe medium successfully (FIG. 3).

It is possible to develop various embodiments of the inventiveproduction method of sol-gel and use of sol-gel for enrichment ofphosphopeptide, it cannot be limited to examples disclosed herein and itis essentially according to claims.

1. A production method of sol-gel (100) wherein the steps comprising of:adding one or more of tantalum (V) alkoxide, tantalum (V) methoxide,tantalum (V) ethoxide, tantalum (V) propoxide, tantalum (V) isopropoxideand tantalum (V) butoxide into the mixture (101); performing a mixingprocess during addition of material (102); adding an acid solution suchas HNO₃, HCl, H₂SO₄, CH₃COOH, CF₃COOH etc. during the mixing process(103); decomposing the sol-gel with tantalum content through acentrifuge process (104).
 2. A production method of sol-gel (100)according to claim 1, wherein before the addition of tantalum metal intothe mixture (101), the solution comprising polyethylene glycol (PEG)(Mr-600-3000) in a ratio of 30-70% by volume is diluted with ethanol:water mixture (18:0.5-8:3.0, v/v) (1%, v/v).
 3. A production method ofsol-gel (100) according to claim 1, wherein while the mixing processcontinues, up to 0.1-0.5% of a 100% fuming HNO₃ solution is added intothe synthesis medium (103).
 4. A production method of sol-gel (100)according to claim 1, wherein the sol-gel with tantalum content obtainedis washed and then taken into its final form after being dried.
 5. Aproduction method of sol-gel (100) according to claim 4, wherein thesol-gel with tantalum content is washed with solvents such as ethanol,water and methanol.
 6. A production method of sol-gel (100) according toclaim 4, wherein the drying process is carried out at temperature of 40°C. in a vacuum environment between 2-200 mbars.
 7. A method (200)according to claim 1, wherein the method of phosphopeptide enrichment(200) which is realized using the sol-gel obtained comprises steps of:inserting the sol-gel comprising the tantalum metal into the syringe(201); adding some acid into the peptide mixture and adjusting PH (202);drawing a certain amount of the solution generated into the syringe andreleasing it (203); carrying out the washing process (204); andwithdrawing the phosphopeptides from the syringe (205).
 8. A method(200) according to claim 7, wherein after the sol-gel comprising thetantalum metal is inserted the into the syringe (201), firstly mixtureof acetonitrile:water:trifluoroacetic acid (1:1:0.005-0.3, v/v/v) isadded into the peptide mixture obtained as a result of enzymaticdigestion and the PH is adjusted (202).
 9. A method (200) according toclaim 8, wherein in a preferred embodiment, 50 μL of the solutiongenerated is drawn into the syringe and released (203).
 10. A method(200) according to claim 9, wherein the process is repeated for 2-6times.
 11. A method (200) according to claim 7, wherein in order toremove impurities and peptides except phosphopeptides, the mixture ofacetonitrile:water:trifluoroacetic acid (1:1:0.005-0.3, v/v/v) is drawninto the syringe and released.
 12. A method (200) according to claim 11,wherein the process is repeated for 2-6 times.
 13. A method (200)according to claim 7, wherein the solution of 10-100 mM ammoniumbicarbonate comprising ammonia in the washing process is pulled throughthe syringe (204) lastly and the phosphopeptides are withdrawn from thesyringe (205).